Liposomes as contrast agents for ultrasonic imaging

ABSTRACT

Liposomes suitable as ultrasound contrast agents which contain media of various types including gases, gaseous precursors activated by pH, temperature or pressure, as well as other solid or liquid contrast enhancing agents, are described. Methods of using the same as ultrasound contrast agents are also disclosed. The present invention also comprises novel methods for synthesizing liposomes having encapsulated therein gases.

RELATED APPLICATION

This application is a divisional of U.S. Ser. No. 163,039, filed Dec. 6,1993, which in turn is a divisional of U.S. Ser. No. 085,608, filed Jun.30, 1993, now U.S. Pat. No. 5,344,381, which in turn is a divisional ofU.S. Ser. No. 018,112, filed Feb. 17, 1993, now abandoned, which in turnis a divisional of U.S. Ser. No. 967,974, filed Oct. 27, 1992, now U.S.Pat. No. 5,352,435, which in turn is a divisional of U.S. Ser. No.818,069, filed Jan. 8, 1992, now U.S. Pat. No. 5,230,882, which in turnis a divisional of U.S. Ser. No. 750,877, filed Aug. 26, 1991, now U.S.Pat. No. 5,123,414, which in turn is a divisional of U.S. Ser. No.569,828, filed Aug. 20, 1990, now U.S. Pat. No. 5,088,499, which in turnis a continuation-in-part of application U.S. Ser. No. 455,707, filedDec. 22, 1989, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of ultrasonic imaging, and, morespecifically, to the use of liposomes in ultrasonic imaging procedures.

2. Background of the Invention

There are a variety of imaging techniques which have been used to detectand diagnose disease in animals and humans. One of the first techniquesused for diagnostic imaging was X-rays. The images obtained through thistechnique reflect the electron density of the object being imaged.Contrast agents such as barium or iodine are used to attenuate or blockX-rays such that the contrast between various structures is increased.For example, barium is used for gastrointestinal studies to define thebowel lumen and visualize the mucosal surfaces of the bowel. Iodinatedcontrast media is used intravascularly to visualize the arteries andthis is called angiography. X-rays, however, are known to be dangerous.The radiation employed in X-rays is ionizing and the deleterious effectsof the ionizing radiation are cumulative.

Magnetic resonance imaging (MRI) is another important imaging technique,however it has the drawbacks of expense and the fact that it cannot beconducted as a portable examination. In addition, MRI is not availableat many medical centers.

Radionuclides, employed in nuclear medicine, provide another imagingtechnique. In employing this technique, radionuclides such as technetiumlabelled compounds are injected into the patient, and images areobtained from gamma cameras. Nuclear medicine techniques, however,suffer from poor spatial resolution and expose the animal or patient tothe deleterious effects of radiation. Furthermore, there is a problemwith the handling and disposal of radionuclides.

Ultrasound, another diagnostic imaging technique, is unlike nuclearmedicine and X-rays in that it does not expose the patient to theharmful effects of ionizing radiation. Moreover, unlike magneticresonance imaging, ultrasound is relatively inexpensive and can beconducted as a portable examination. In using the ultrasound technique,sound is transmitted into a patient or animal via a transducer. When thesound waves propagate through the body, they encounter interfaces fromtissues and fluids. Depending on the reflectivity and acousticproperties of the tissues and fluids in the body, the ultrasound soundwaves are either reflected or absorbed. When sound waves are reflectedby an interface they are detected by the receiver in the transducer andprocessed to form an image. The acoustic properties of the tissues andfluids within the body determine the contrast which appears in theresultant image.

Advances have been made in recent years in ultrasound technology.However, despite these various technological improvements, ultrasound isstill an imperfect tool in a number of respects, particularly Withrespect to the detection of disease in the liver and spleen, kidneys andvasculature and in measuring blood flow. The ability to detect andmeasure these things depends on the difference in acoustic propertiesbetween blood or other tissues and the surrounding tissues. As a result,contrast agents have been sought which will increase the acousticdifference between blood and surrounding tissues in order to improve themeasurement of blood flow, or between one tissue and another such asbetween the liver and a tumor in order to improve disease detection.

The principles underlying image formation in ultrasound have directedresearchers to this pursuit of contrast agents. When sound waves fromultrasound pass through a substance, the acoustic properties of thatsubstance will depend upon the velocity of the sound and the density ofthat substance. Changes in the acoustic properties or acoustic impedanceof the substance are most pronounced at interfaces of differentsubstances with greatly different density or acoustic impedance,particularly at the interface between solids, liquids and gases. Whenthe ultrasound sound waves encounter such interfaces, the changes inacoustic impedance result in a more intense reflection of sound wavesand a more intense signal in the ultrasound image.

Many of the prior art contrast agents developed to date for ultrasoundhave comprised liquids containing microbubbles of gas where themicrobubbles have been encapsulated with gelatin or saccharine. Thosemicrobubble and gelatin/saccharine constructs have most often beenprepared using agitation techniques. Other prior art is directed toattempts with protein-associated air bubbles or air bubbles incorporatedin microspheres composed of either albumin or collagen. Furthermore,heavy metal particulates have been evaluated as ultrasound contrastagents. There have also been some reports of liposomes described asuseful in ultrasonic applications having gas or gaseous precursorsencapsulated therein.

While the prior art has produced some ultrasound contrast agents whichare echogenic on ultrasound, that is, provide a contrast enhancementsignal, the contrast agents developed thus far have various problems.The protein based air bubble systems have the drawback that a foreignprotein which may be antigenic and potentially toxic is being employed.The liposomal contrast agents have had problems with uneven sizedistribution and poor stability. The gaseous precursor containingliposomes have also been inefficient in their ability to form contrastenhancing gas in vivo. Moreover, while some of the prototype prior artcontrast agents have demonstrated echogenic effects as transpulmonaryvascular contrast agents, many of these agents have failed todemonstrate a convincing effect on improving tumor imaging in, forexample, the liver or spleen. Furthermore, many of the methods forpreparing these ultrasound contrast agents, particularly the gasencapsulated liposomes, are inefficient, expensive, and otherwiseunsatisfactory.

The present invention is directed to answering these and other importantneeds.

SUMMARY OF THE INVENTION

In one embodiment, the present invention is directed to a contrast agentfor ultrasonic imaging comprising an ionophore-containing liposomehaving encapsulated therein a pH-activated gaseous precursor.

In another embodiment, the present invention is directed to a contrastagent for ultrasonic imaging which comprises a liposome havingencapsulated therein a photo-activated gaseous precursor.

In a third embodiment, the present invention is directed to a contrastagent for ultrasonic imaging which comprises a liposome havingencapsulated therein a temperature-activated gaseous precursor.

In a further embodiment, the present invention is directed to a contrastagent for ultrasonic imaging which comprises a liposome havingencapsulated therein a solid or liquid contrast enhancing agent.

An even further embodiment of the invention is directed to a method forimaging a patient using ultrasound comprising administering to thepatient a liposome of the invention and scanning the patient usingultrasound.

In a still further embodiment, the present invention comprises novelmethods for encapsulating a gas within the internal space of a liposometo produce contrast agents for ultrasonic imaging.

The contrast agents embodied within the present invention are echogenic,that is, capable of reflecting ultrasound waves to enhance signalintensity on an ultrasound image. In certain preparations particularlydesigned as intravascular contrast agents, the present contrast agentsare small enough to pass through the capillaries of pulmonarycirculation and are effective in providing good contrast enhancement ofthe heart, arterial system and venous system. In other preparationsdesigned for injecting into other structures or cavities, the vesiclesare larger to maximize echogenicity and provide highly effectivecontrast enhancement. In accordance with the present invention, theliposomes with the gas, gaseous precursors and/or solid or liquidcontrast enhancing agents encapsulated therein can be produced indefined and reproducible sizes. The present invention also allowstargeting and delivery of the contrast agent to specific sites such asthe vasculature, liver, spleen and kidney. The present invention is freefrom the toxicity associated with the use of foreign proteins toencapsulate air bubbles and also minimizes the likelihood of embolismsoccurring. In addition, the liposomes of the present invention arecapable of long term storage. Moreover, the novel methods of theinvention for encapsulating gas within the internal space of a liposomeare highly efficient and inexpensive to carry out.

The liposomal ultrasound contrast agents of the invention permitadvanced imaging of organs and tissues in a way not previouslycontemplated. Because liposome membranes can be optimized for blood poolor circulation half-life, effective perfusion and blood pool contrastagents will be available. This will be useful in the heart, for example,for diagnosing ischemia and in other organs for diagnosing decreasedblood flow or shunts. In blood pool lesions such as cavernoushemangioma, these agents will be useful for making accurate diagnoses.Because these agents can be optimized for uptake by healthy cells inorgans such as the liver and spleen, the contrast agent facilitates theultrasonic detection and characterization of tumors in these organs.

These and other features of the invention and the advantages thereofwill be further described in the drawings and description below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. In this figure, the general method in which a gaseous precursorreacts to form a gas in response to a change in pH within a vesicle isdescribed. In the example illustrated, bicarbonate salts are entrappedwithin the interior aqueous space of the vesicle and an ionophore suchas, for example, p-trifluoromethoxy-carbonylcyanide phenylhydrazone ispresent within the liposome membrane matrix to promote hydrogen ion fluxacross the liposomal membrane. The pH change within the vesicle interiorfacilitated by the ionophore results in the formation of a highlyechogenic carbon dioxide gas.

FIG. 1B. In this figure, the general method in which a gaseousprecursors reacts to form gases upon exposure to UV light is described.In the specific example illustrated, diazonium compounds trapped insidethe lipid vesicles form a highly echogenie nitrogen gas as a result ofUV exposure.

FIG. 1C. In this figure, the general method in which a gaseous precursorforms a gas in response to an increase in temperature, is illustrated.Once injected into a patient, methylactate, for example, is transformedfrom a liquid to a highly echogenic gas as a result of the increase intemperature from room temperature to physiological temperature.

FIG. 1D. In this figure, one method of entrapping a particulate solidcontrast enhancing agent such as magnetite, within a liposome isdescribed. In the illustrated method, a mixture of ferrous and ferricsalts is entrapped within the aqueous core of the liposome. An ionophoresuch as valinomycin is incorporated within the matrix of the liposome inorder to increase the rate of proton flux across the membrane. Prior toor during use, the pH on the exterior of the vesicle is then increasedby the addition of the appropriate alkali resulting in an increase inthe pH in the interior of the liposome. The increase in pH in turnpromotes base catalysis which results in the in situ formation of highlyechogenic magnetite within the liposome. It is equally possible toentrap preformed solid contrast enhancing agents such as preformedmagnetite in the liposomes.

FIG. 2. In this figure is illustrated one method of modifying thesurface of a liposome with polymer such as polyethylene glycol (PEG), soas to be able to modulate the clearance and biodistribution kinetics ofthe liposomes with entrapped gas, gaseous precursors and/or solid orliquid contrast enhancing agents.

FIG. 3. This figure illustrates the basic pressurization anddepressurization phenomenons behind some of the devices and methods forpreparing the gas-containing liposomes of the invention. First,liposomes are added to a vessel, and the vessel is then pressurized withgas. Under pressure, the gas goes into solution and passes across theliposome membranes. When the pressure is released, gas bubbles formwithin the liposomes.

FIG. 4. This figure illustrates one apparatus of the invention forsynthesizing liposomes having encapsulated therein a gas. The apparatusis utilized by placing a liquid media which contains liposomes into thevessel. A cap is then threaded onto the vessel opening providing apressure tight seal. The vessel is pressurized by fitting a cartridgecontaining a gas, such as carbon dioxide, into an inlet port. Thecartridge discharges its contents into the upper end of a tube fittedinto the vessel. The gas flows through the tube and exits at the lowerend of the tube into the bottom of the vessel. After the gas has beenintroduced into the vessel, the vessel can then be depressurized byejecting the liquid from the vessel.

FIG. 5. This figure illustrates another apparatus of the invention forsynthesizing liposomes having encapsulated therein a gas. A syringe inwhich liposomes have been placed is connected via one or more filters ofvarious pore sizes to an inlet/outlet port and valve of the pressurevessel. The syringe is then emptied through the filters and theinlet/outlet port and valve into the bottom of the vessel.Alternatively, the vessel may be directly loaded with the liposomeswithout using the syringe and/or filters, and/or inlet/outlet port andvalve. The vessel is then pressurized with a gas, resulting in agas-containing liposome composition. The gas-containing liposomecontents of the vessel may then be discharged through the inlet/outletport and valve and the filter assembly, and emptied into the syringe.Alternatively, the liposome may be removed directly without passingthrough the filter and/or inlet/outlet port and valve and/or emptyinginto the syringe.

FIG. 6. This figure illustrates a further apparatus of the invention forsynthesizing liposomes having encapsulated therein a gas. The gas entersa vessel in which a liquid media containing liposomes has been placedthrough an inlet port, flows through a tube and discharges into thebottom of the vessel. From the bottom of the vessel, the gas bubblesupward through the liquid. Depressurization is accomplished by opening avalve on a separate outlet port, thereby ejecting the liquid from thebottom of the vessel through the tube.

FIG. 7. This figure illustrates another apparatus for synthesizingliposomes having encapsulated therein a gas. As this figure illustrates,the apparatus required to practice the method of the invention need onlybe a simple vessel with a port for introducing and discharging thepressurized gas and liposomes.

FIG. 8. This figure illustrates another apparatus for synthesizingliposomes having encapsulated therein a gas. This apparatus is utilizedby placing a liquid media which contains liposomes in a vessel jacketedby a chamber through which a coolant circulates. A high frequency soundwave generator is attached to the vessel. In use, the vessel ispressurized using a gas introduced through a gas port. The sound wavegenerator transforms electrical energy into mechanical oscillations andtransmits the oscillations into the liquid through a horn which extendsinto the vessel. As the liposomes break up and reform as a result of themechanical forces, they encapsulate the dissolved gas within theirinternal aqueous core. Following sonication, the vessel is depressurizedand the encapsulated gas forms bubbles, thereby transforming theliposomes into gas-containing liposomes.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention provides a contrast agent forultrasound imaging which comprises an ionophore-containing liposomehaving encapsulated therein a pH-activated gaseous precursor.

As used herein, the phrase "ionophore-containing liposome" denotes aliposome having incorporated in the membrane thereof an ionophore. Theterm "ionophore", as used herein, denotes compounds which are capable offacilitating the transport of hydrogen ions or hydroxide ions across theliposome membrane to effect a change in pH inside the liposome membrane,and include compounds commonly referred to as proton carriers andchannel formers. Suitable ionophores include proton carriers such asnitro-, halo- and oxygenated phenols and carbonylcyanidephenylhydrazones. Preferred of such proton carriers are carbonylcyanide,p-trifluoromethoxyphenylhydrazone (FCCP), carbonylcyanideM-chlorophenylhydrazone (CCCP), carbonylcyanide phenylhydrazine (CCP),tetrachloro-2-trifluoromethyl benzimidazole (TTFB),5,6-dichloro-2-trifluoromethyl benzimidazole (DTFB), and Uncoupler 1799Suitable channel formers include gramicidin, alamethicin, filipin,etruscomycin, nystatin, pimaricin, and amphotericin. Other suitableproton carriers include the following compounds which preferably exhibitselectivity for cations, but will also transport protons and/orhydroxide ions: valinomycin, enniatin (type A, B or C), beauvericin,monomycin, nonactin, monactin, dinactin, trinactin, tetranactin,antamanide, nigericin, monensin, salinomycin, narisin, mutalomycin,carriomycin, dianemycin, septamycin, A-204 A, X-206, X-537 A(lasalocid), A-23187 and dicyclohexyl-18-crown-6. Such ionophores arewell known in the art and are described, for example in Jain et al.,Introduction to Biological Membranes, (J. Wiley and Sons, N.Y. 1980),especially pp. 192-231, and Methyl Ions In Biological Systems, ed. H.Sygel, Vol. 19, "Antibiotics And Their Complexes" (Dekker, N.Y. 1985),disclosures of each of which are incorporated herein by reference intheir entirety. The ionophores may be used alone or in combination withone another.

It has been found that although liposomes are not impermeable to protonsor hydroxide ions, the permeability coefficient of liposomes isgenerally so very low that it often takes weeks or months to dissipate apH gradient. Providing a more rapid transport of hydrogen ions orhydroxide ions across a liposome membrane in order to activatepH-modulated gaseous precursors is necessary. The incorporation ofionophores in the liposome membrane, in accordance with the presentinvention, provides the necessary means of transporting the activatingions. By increasing the rate of hydrogen or hydroxide ion flux acrossthe liposome membrane, such ionophores will increase the rate within theliposome of gas formation from the pH-activated gaseous precursor. Thisphenomenon is diagrammatically represented in FIG. 1A.

The phrase "pH-activated gaseous precursor", as used herein, denotes acompound in solid or liquid form which, when exposed to a drop in pH,will form a gas. As noted above, this concept is illustrated in FIG. 1A.Such compounds include, but are not limited to, metal carbonate andbicarbonate salts, such as the alkali metal carbonates and bicarbonates,and the alkaline earth carbonates and bicarbonates, and mixturesthereof. Exemplary of such compounds are lithium carbonate, sodiumcarbonate, potassium carbonate, lithium bicarbonate, sodium bicarbonate,potassium bicarbonate, magnesium carbonate, calcium carbonate, magnesiumbicarbonate, and the like. Also useful gas generating compounds areammonium carbonate, ammonium bicarbonate, ammonium sesquecarbonate,sodium sesquecarbonate, and the like. These compounds, when dissolved inwater, show a pH of greater than about 7, usually between about 8 andabout 12. Other pH-activated gaseous precursors include aminomalonate,which, when dissolved in water, generally shows a pH of about 5 to 6.The pka1 of aminomalonate is 3.32 and the pka2 is 9.83. Aminomalonate iswell known in the art, and its preparation is described, for example, inThanassi, Biochemistry, Vol. 9, no. 3, pp. 525-532 (1970), Fitzpatricket al., Inorganic Chemistry, Vol. 13, no. 3, pp. 568-574 (1974),Stelmashok et al., Koordinatsionnaya Khimiya, Vol. 3, no. 4, pp. 524-527(1977). Other suitable pH-activated gaseous precursors will be apparentto those skilled in the art.

As those skilled in the art would recognize, such compounds can beactivated prior to administration, if desired. Of course, by choosing agaseous precursor with the appropriate pKa, one skilled in the art canprepare a liposome whereby gas will form in the liposome afterintravenous injection or injection into a body cavity. Even whenexposure to the appropriate pH occurs prior to administration, anadvantage is achieved in that the liposome with the gaseous precursor isa more stable entity than a liposome which has been placed on the shelfwith a gas encapsulated therein. Accordingly, greater shelf life isevident from the use of liposomes which encapsulate a pH-activatedgaseous precursor. It has also been discovered that the use ofionophores allows liposomes entrapping pH-activated gaseous precursorsto efficiently produce gas when exposed to a pH gradient. The resultinggas-containing liposomes are capable of being detected easily in vivobecause of their lower density as compared to the surrounding bodilystructures and organs.

In a second embodiment of the invention, a contrast agent for ultrasonicimaging is provided which comprises a liposome having encapsulatedtherein a photo-activated gaseous precursor. As used herein, the phrase"photo-activated gaseous precursor" denotes a light sensitive chemicalwhich forms a gas after exposure to such light. This concept isillustrated in FIG. 1B. Suitable photosensitive compounds includediazonium compounds which decompose to form nitrogen gas after exposureto ultraviolet light. Another suitable compound is aminomalonate. As oneskilled in the art would recognize, other gaseous precursors may bechosen which form gas after exposure to light. Depending upon theapplication, exposure to such light may be necessary prior to in vivoadministration, or in some instances can occur subsequent to in vivoadministration. Even when exposure to the appropriate light occurs priorto administration, an advantage is achieved in that the liposome withthe gaseous precursor is a more stable entity than a liposome which hasbeen placed on the shelf with a gas encapsulated therein. Accordingly,greater shelf life is evident from the use of the liposome whichencapsulates a photo-activated gaseous precursor. The resultinggas-containing liposomes are capable of being detected easily in vivobecause of their lower density as compared to the surrounding bodilystructures and organs.

In a third embodiment, the present invention is directed to a contrastagent for ultrasonic imaging which comprises a liposome havingencapsulated therein a temperature-activated gaseous precursor. As usedherein, the phrase "temperature-activated gaseous precursor" denotes acompound which forms a gas following a change in temperature. Thisconcept is illustrated in FIG. 1C. Suitable temperature-activatedgaseous precursors are well known to those skilled in the art, andinclude, for example, methylactate, a compound which is in a liquidphase at ambient temperatures, but which forms a gas at physiologicaltemperatures. As those skilled in the art would recognize, suchcompounds can be activated prior to administration or, as in the case ofmethylactate, can be activated upon injection into the patient. Evenwhen exposure to the appropriate temperature occurs prior toadministration, an advantage is achieved in that the liposome with thegaseous precursor is a more stable entity than a liposome which has beenplaced on the shelf with a gas encapsulated therein. Accordingly,greater shelf life is evident from the use of the liposome whichencapsulates a temperature-activated gaseous precursor. The resultinggas-containing liposomes are capable of being detected easily in vivobecause of their lower density as compared to the surrounding bodilystructures and organs. In addition, as those skilled in the art wouldrecognize, such temperature sensitive gas-forming liposomes can be usedas indicators of in vivo temperature.

Liposomes encapsulating solid and liquid contrast enhancing agents arealso encompassed within the subject invention. As used herein, the terms"solid contrast enhancing agent" and "liquid contrast enhancing agent",denotes solid particulate materials, and solubilized or liquidmaterials, respectively, which are echogenic on ultrasound. Suitablesolid contrast enhancing agents will be readily apparent to thoseskilled in the art once armed with the present disclosure, and includemagnetite (Fe₃ O₄), solid iodine particles such as particles formed fromiodipamide ethyl ester, and particles formed by precipitating a waterinsoluble derivative of the ionic iodinated contrast medium metricate.Suitable liquid contrast enhancing agents will be readily apparent tothose skilled in the art, once armed with the present disclosure, andinclude solubilized iodinated contrast agents. The latter is preferablyused as an intravascular contrast agent for the purpose of visualizingflow, but is also highly effective for detecting tumors in the liver andspleen.

As those skilled in the art will recognize upon reading the presentdisclosure, some solid and liquid contrast enhancing agents can beformed in situ. In the case of magnetite, for example, iron salts can beencapsulated at low pH (e.g., pH 2) and the external pH of the outsidesolution raised. The iron oxides then precipitate within the vesicleforming magnetite. To facilitate the transport of hydroxide ion into thevesicle, an ionophore such as valinomycin is incorporated into theliposome membrane. This is similar to the situation shown in FIG. 1D,except that in this instance, pH is being raised, rather than lowered.

The liposomes employed in the present invention can be prepared usingany one of a variety of conventional liposome preparatory techniques. Aswill be readily apparent to those skilled in the art, such conventionaltechniques include sonication, chelate dialysis, homogenization, solventinfusion coupled with extrusion, freeze-thaw extrusion,microemulsification, as well as others. These techniques, as well asothers, are discussed, for example, in U.S. Pat. No. 4,728,578, U.K.Patent Application G.B. 2193095 A, U.S. Pat. No. 4,728,575, U.S. Pat.No. 4,737,323, International Application PCT/US85/01161, Mayer et al.,Biochimica et Biophysica Acta, Vol. 858, pp. 161-168 (1986), Hope etal., Biochimica et Biophysica Acta, Vol. 812, pp. 55-65 (1985), U.S.Pat. No. 4,533,254, Mahew et al., Methods In Enzymology, Vol. 149, pp.64-77 (1987), Mahew et al., Biochimica et Biophysica Acta, Vol. 75, pp.169-174 (1984), and Cheng et al., Investigative Radiology, Vol. 22, pp.47-55 (1987), and U.S. Ser. No. 428,339, filed Oct. 27, 1989. Thedisclosures of each of the foregoing patents, publications and patentapplications are incorporated by reference herein, in their entirety. Asa preferred technique, a solvent free system similar to that describedin International Application PCT/US85/01161, or U.S. Ser. No. 428,339,filed Oct. 27, 1989, is employed in preparing the liposomeconstructions. By following these procedures, one is able to prepareliposomes having encapsulated therein a gaseous precursor or a solid orliquid contrast enhancing agent.

The materials which may be utilized in preparing the liposomes of thepresent invention include any of the materials or combinations thereofknown to those skilled in the art as suitable in liposome construction.The lipids used may be of either natural or synthetic origin. Suchmaterials include, but are not limited to, lipids such as cholesterol,phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylglycerol, phosphatidicacid, phosphatidylinositol,lysolipids, fatty acids, sphingomyelin, glycosphingolipids, glucolipids,glycolipids, sulphatides, lipids with ether and ester-linked fattyacids, polymerizable lipids, and combinations thereof. As one skilled inthe art will recognize, the liposomes may be synthesized in the absenceor presence of incorporated glycolipid, complex carbohydrate, protein orsynthetic polymer, using conventional procedures. The surface of aliposome may also be modified with a polymer, such as, for example, withpolyethylene glycol (PEG), using procedures readily apparent to thoseskilled in the art. This is illustrated in FIG. 2. Any species of lipidmay be used, with the sole proviso that the lipid or combination oflipids and associated materials incorporated within the lipid matrixshould form a bilayer phase under physiologically relevant conditions.As one skilled in the art will recognize, the composition of theliposomes may be altered to modulate the biodistribution and clearanceproperties of the resulting liposomes.

To incorporate ionophores into the liposome membrane, the ionophores,which are lipophilic, are simply added to the lipid mixture, and theliposomes are prepared in the usual fashion.

In addition, the size of the vesicles can be adjusted by a variety ofprocedures including filtration, sonication, homogenization and similarmethods to modulate liposomal biodistribution and clearance. To increaseinternal aqueous trap volume, the vesicles can be subjected to repeatedcycles of freezing and thawing.

The liposomes of the invention may be of varying sizes, but preferablyhave a mean outer diameter between about 30 nanometers and about 10microns. As is known to those skilled in the art, vesicle sizeinfluences biodistribution and, therefore, different size vesicles areselected for various purposes. For intravascular use, for example,vesicle size is generally no larger than about 2 microns, and generallyno smaller than about 30 nanometers, in mean outer diameter. In the sizerange of 2-3 microns, the vesicles are by their nature multilamellar.Within this range, to maximize echogenicity with a liposomal contrastagent which has a short intravascular half life, larger vesicles areselected, e.g., about 1 to about 2 microns in mean outer diameter. Forsustained blood pool imaging such as for perfusion, smaller vesicles areused, e.g., between about 100 nanometers and several hundred nanometersin mean outer diameter. To provide ultrasound enhancement of organs suchas the liver and to allow differentiation of tumor from normal tissuesmaller vesicles between about 30 nm and about 100 nm in mean outerdiameter which will cross the capillary fenestrations into the liver andincrease the uptake by liver may be employed. For imaging of bodycavities and non-vascular injection, larger vesicles, e.g., betweenabout 2 and about 10 micron mean outside diameter may be employed tomaximize echogenicity of entrapped air.

The lipids employed in the preparations are selected to optimize theparticular diagnostic use, minimize toxicity and maximize shelf-life ofthe product. Neutral vesicles composed of phosphatidylcholine andcholesterol function quite well as intravascular contrast agents toentrap gas, magnetite, solid iodine particles and solubilized iodinatedcontrast agents. To improve uptake by cells such as thereticuloendothelial system (RES), a negatively charged lipid such asphosphatidylglycerol, phosphatidylserine or similar materials is added.To prolong the blood pool half life, highly saturated lipids which arein the gel state at physiological temperature such asdipalmitoylphosphatidylcholine are used. For even greater vesiclestability and prolongation of blood pool half-life the liposome can bepolymerized using polymerizable lipids, or the surface of the vesiclecan be coated with polymers such as polyethylene glycol so as to protectthe surface of the vesicle from serum proteins, or gangliosides such asGM1 can be incorporated within the lipid matrix.

The pH-activated gaseous precursor, the photo-activated gaseousprecursor, the temperature-activated gaseous precursor, and/or the solidor liquid contrast enhancing agent can be incorporated into the liposomeby being added to the medium in which the liposome is being formed, inaccordance with conventional protocol.

The liposomes of the present invention are useful in ultrasound imaging.

In a still further embodiment, the present invention comprises noveldevices and methods for encapsulating a gas within the internal space ofthe liposome. The liposomes thus produced are also useful in ultrasoundimaging.

In general terms, in using the device and carrying out the method of theinvention, liposomes are added to a vessel, and the vessel is thenpressurized with gas. Under pressure, the gas goes into solution andpasses across the liposome membranes. When the pressure is released, gasbubbles form within the liposomes. FIG. 3 illustrates the generalpressurization and depressurization phenomenons of the invention.

In using the pressurization devices and carrying out the pressurizationprocesses of the invention, it is preferable to utilize a liposome thatdoes not contain a sterol as part of the membrane. The presence ofsterols such as cholesterol in the liposome membrane, particularly ifthey are present in significant quantities, i.e., greater than about 5%of the membrane by volume, tends to substantially inhibit the flow ofcertain solubilized gases, such as carbon dioxide, across the liposomemembrane. Also, if the membrane is comprised largely of saturatedlipids, that is greater than about 80% saturated lipids, the flow ofcertain solubilized gases, such as that of carbon dioxide, is alsosubstantially inhibited.

The liposomes may or may not be pre-sized prior to being placed withinthe pressurization vessel. In the case of pre-sizing this may beachieved by a variety of means including, but not limited to,sonication, gel-filtration, filtration through polystyrene orpolycarbonate filters or other filters of suitable material, Frenchpress or microemulsification methods.

There is shown is FIG. 4 an apparatus for synthesizing liposomes havingencapsulated therein a gas. The apparatus is, in essence, a modifiedsoda seltzer bottle. The apparatus is utilized by placing a liquidmedia, such as a phosphate buffered saline solution which containsliposomes, into a vessel (1). Typically, the liposomes will be comprisedof egg phosphatidylcholine, although as described above, other lipidscan be employed in the preparation of the liposomes. A cap (10) is thenthreaded onto the vessel opening (11), providing a pressure tight seal.The vessel is pressurized by fitting a cartridge (9) containing a gas ora combination of gases, such as carbon dioxide, into an inlet port (8).The vessel may be constructed of any suitable material, such as glass oracrylic, and may be disposable, if desired. The cartridge discharges itscontents into the upper end (6) of a tube (3), preferably a polyethylenetube, fitted into the vessel (1). The gas flows through the tube andexits at the lower end (4) of the tube. The gas then bubbles upwardthrough the liquid media so that at least a portion of the gas dissolvesin the liquid media. Generally, the pressure of the gas in this andother pressurization devices and methods disclosed herein, is betweenabout 2 and about 400 psi. In the preferred embodiment, the vessel ispressurized to between the 50-120 psi range. Within this range,generally higher pressures are preferred for certain gases, such asnitrogen, and gradually lower pressures required for others, such ascarbon dioxide. If necessary, additional gas cartridges may be used. Apressure gauge (29) indicates the pressure in the vessel.

The liposome membranes are permeable to the pressurized gas. Thus, asthe gas bubbles through the liquid media, a portion of the dissolved gasis encapsulated within the internal aqueous environment of theliposomes. To enhance the dissolving of the gas into the liquid, it isdesirable to promote mixing of the gas and the liquid, and bubbling thegas through the liquid assists in this. In the preferred embodiment,this mixing is further enhanced by providing the vessel with a convexshaped bottom (2) projecting into the vessel. The lower end of the tubedischarges the gas near the most inward point (5) on the convex shapedbottom.

After the gas has been introduced into the vessel (1), the vessel isdepressurized by ejecting the liquid therefrom. Ejection is accomplishedby actuating a discharge lever (12), in the cap (10). Actuation of thedischarge lever opens an outlet port (7) so that the gas pressure forcesthe liquid to enter the tube (3) at its lower end (4), flow up the tubeand out of the vessel through the discharge port. Forcing the liquidthrough the tube promotes further mixing of the gas and liquid. Upondepressurization, the dissolved gas encapsulated by the liposomes comesout of solution and forms bubbles within the liposomes, thereby formingliposomes having encapsulated therein a gas.

Alternatively, the method described above could be practiced using theapparatus shown in FIG. 5. If desired, the liposomes may be pre-sized byinjecting them from a syringe (33) through one or more filter(s) (30),through inlet/outlet port (35) and valve (39), and then through tube(38) into vessel (34). Alternatively, the liposomes are simply placed ina vessel (34). Vessel (34) is equipped with valves (39), inlet/outletport (35), inlet/outlet port (36) and tube (38). The vessel (34) isconstructed so that it can be pressurized with a gas or combination ofgases such as carbon dioxide, oxygen, nitrogen, xenon, argon, neon andhelium by means of a valve or inlet port (36) and external pressuresource (37) which can be a gas line, tank or disposable cartridge. Thevalve(s) (39) may be constructed so as to be able to vent excesspressure without dispensing the liposomes. The vessel may be constructedof any suitable material, such as glass or acrylic, and may bedisposable, if desired.

In use, the vessel (34) is first loaded with a liposomal containingsolution, using if desired, syringe (33), with or without filter (30).The vessel is then pressurized with gas using external pressure source(37) which passes gas through inlet port (36) through valve (39) andtube (38) into vessel (34). Under pressure, the gas goes into solutionand passes across the liposome membranes. When the pressure is released,gas bubbles form within the liposomes. The pressurized vessel (34) hasan inlet/outlet port (35) to which one or more filters (30) may beattached. In FIG. 5, the use of Luer lock fittings (31) and (32) isillustrated as an example of a means for connecting the pressure vessel(34), filters (30) and syringe (33). However, any suitable means ofcoupling the devices may be employed. The pore size of suitable filtersmay vary widely between about 0.015 micron and about 10 microns. Morethan one filter, if desired, may be employed in serial connection. Thefunction of the filter(s) (30) is threefold: to promote decavitation ofany bubbles formed external to the liposomes after preparation of thegas-encapsulating liposomes; to promote sizing of the liposomes eitherbefore or after gas-encapsulation; and to remove non-liposomal solidsfrom the suspension. The filter(s) (30) may in turn be connected to asyringe (33). When the vessel is filled with liposomes, they may bedirected from the syringe (33) through the filter(s) (30) and throughthe inlet/outlet port (35) and valve (39) into pressure vessel (34). Inaddition, when the contents of vessel (34) are released by means of theoutlet valve (35) of the vessel, the output stream may be directedthrough the filters (30) into the syringe (33). If desired, the vessel(34) may be directly loaded and unloaded without passing throughinlet/outlet port (35) and valve (39), filter (30) and/or syringe (33).The advantage of this procedure is that the device can be pre-packagedas a stand-alone, sterile unit ready for use. It should also be notedthat it is not necessary that either the filters or syringe be used asdescribed, e.g., the output stream of the device may be directed into aseparate container prior to being taken up, for example, into a syringefor injection.

The method discussed above could also be practiced using the apparatusshown in FIG. 6, wherein the gas enters vessel (13) in which a liquidmedia containing liposomes has been placed, through inlet port (14),flows through tube (15) and discharges into the bottom of the vessel.The vessel may be constructed of any suitable material, such as glass oracrylic, and may be disposable, if desired. From the bottom of thevessel, the gas bubbles upward through the liquid. Depressurization isaccomplished by opening a valve, not shown, on the outlet port (16),thereby ejecting the liquid from the bottom of the vessel through tube(17).

Referring to FIG. 7, the apparatus required to practice such a methodneed only be a simple vessel (27) with a port (28) for introducing theliposomes and the pressurized gas and discharging the same. The vesselmay be constructed of any suitable material, such as glass or acrylic,and may be disposable, if desired.

The inventors have discovered that with some gases, such as with carbondioxide, it is helpful to bubble the gas through the liquid in order todissolve the gas. Also, with some gases, such as with nitrogen, it ishelpful to cool the liquid to approximately the 1°-4° C. range. Incarrying out the pressurization, pressures between about 2 and about 400psi, preferably between about 30 and about 100 psi should be employed.The inventors have also discovered that it is preferable that thedepressurization occur quickly over several seconds or less.

It should be noted that the method described above is particularlyadapted for use with liposomes having membranes which are relativelypermeable to the gas. However, the inventors have found that bysubjecting the liposome-containing liquid media to high frequency soundwaves, as discussed below, even relatively impermeable membranecompositions can be easily utilized. As a general rule and as notedabove, membranes composed of significant amounts of sterols or composedlargely of saturated lipids are relatively impermeable to certain gasessuch as carbon dioxide, however, egg phosphatidyl choline, for example,is highly permeable.

There is shown in FIG. 8 an apparatus for synthesizing liposomescontaining gas which uses sonication. The apparatus is utilized byplacing a liquid media containing liposomes, in a vessel (18). A highfrequency sound wave generator (25), which may be a Sonicator (cr),available from Heat Systems-Ultrasonics, Inc., is attached to the vesselusing mating threads (24) formed in the inlet to the vessel and theoutside of the generator. The vessel is then pressurized using a gasintroduced through gas port (22). In the preferred embodiment, thevessel is pressurized to between about 30 and about 120 psi using a gas,such as carbon dioxide, cooled into approximately the 1°-14° C. range,thereby promoting the dissolving of the gas into the liquid media. Thevessel is jacketed by a chamber (19), through which a liquid or gaseouscoolant (26) circulates via inlet and outlet ports (20), (21)respectively, so as to maintain the temperature of the liquid preferablybetween about 1° C. and about 4° C. range during the sonication processdescribed below.

The sound wave generator (25) transforms electrical energy intomechanical energy, at a frequency of approximately 20 kHz and anamplitude approximately in the range of 30-120 μm, by oscillatingpiezoelectric crystals. These oscillations are transmitted and focusedinto the liquid through a horn (23) which extends into the vessel (18),causing high frequency sound waves to propagate through the liquid. Thesound waves cause cavitation in the liquid, i.e., the high frequencyformation and collapse of microscopic bubbles. The process of inducingcavitation by high frequency sound waves is referred to as sonication.The cavitation induces a shearing and tearing action in the liquid,causing the large multilamellar liposomes to tear and reform intosmaller oligolamellar liposomes and eventually, depending on theduration and intensity of the sonication, unilamellar liposomes. As theliposomes break up and reform, they encapsulate the dissolved gas withintheir internal aqueous cores.

Following sonication, the vessel is depressurized and the encapsulatedgas forms bubbles, thereby transforming the liposomes into gascontaining liposomes as before. However, since the gas was introducedinto the liposomes during their break-up caused by the sonication,liposomes having relatively impermeable membranes can be used. By usingsuch membranes, an added advantage in stability is achieved, that is,the gas bubbles do not diffuse back across the membrane as readily aswith other methods and membranes. Thus, the sonication process allowsthe formation of more stable gas containing liposomes. The inventorshave found that generally cholesterol-based liposomal membranes arerelatively impermeable.

Other techniques in addition to sonication can be used to synthesizeliposomes having encapsulated therein a gas, includingmicroemulsification, extrusion, microfluidization, homogenization andthe like, the requirement being that the synthetic process be conductedunder pressurization, preferably at low temperatures.

In each of the foregoing methods for preparing gas-containing liposomes,the liposome preparation is preferably stored under pressure with thegas in solution or the gas pressurization and depressurization processis carried out at or about the time of use.

The liposomes of the present invention and those produced by theapparatus and method of the invention are useful in imaging a patientusing ultrasound. The present invention is useful in imaging a patientgenerally, and/or in specifically diagnosing the presence of diseasedtissue in a patient. The patient can be any type of mammal, but mostpreferably is a human. The method of the invention is particularlyuseful in diagnosing the vasculature, that is, the arterial system, thevenous system and the heart. It is also particularly useful in providingimages of the patient's liver, spleen or kidney.

The imaging process of the present invention may be carried out byadministering a liposome of the invention, that is a liposome selectedfrom the group consisting of an ionophore-containing liposome havingencapsulated therein a pH-activated gaseous precursor, a liposome havingencapsulated therein a photo-activated gaseous precursor, a liposomehaving encapsulated therein a temperature-activated gaseous precursor,and/or a liposome having encapsulated therein a solid or liquid contrastenhancing agent, to a patient, and then scanning the patient usingultrasound imaging to obtain physical images of an internal region of apatient and/or of any diseased tissue in that region. By region of apatient, it is meant the whole patient or a particular area or portionof the patient.

Any of the various types of ultrasound imaging devices can be employedin the practice of the invention, the particular type or model of thedevice not being critical to the method of the invention.

For intravascular use the contrast agent is generally injectedintravenously, but may be injected intra-arterially also. As injectionsare performed, ultrasonic images are obtained with an ultrasoundscanner. In the case of intravascular injection, the liposomal contrastagents generally have a mean outer diameter of smaller than about 2 toabout 3 microns (small enough to pass through the pulmonarycirculation). In other non-vascular applications, the liposomal contrastagent may be injected directly into the area to be scanned, into sitessuch as sinus tracts or the uterine cavity, for example, to assesspatency of the fallopian tubes. In cases of non-vascular injection, theliposomal contrast agent diameter is not constrained by the necessity ofpassing through the pulmonary microvasculature. Therefore largerliposomes can be used to maximize echogenicity.

In administering the liposomes of the present invention, dosage istypically initiated at lower levels and increased until the desiredcontrast enhancement in the patient is achieved. In carrying out themethod of the invention, the liposomes can be used alone, in combinationwith one another, or in combination with other diagnostic and/ortherapeutic agents. Preferable routes of administration will be readilyapparent to those skilled in the art. As those skilled in the art willrecognize, such parameters as dosage and preferable administrationroutes will vary depending upon the age, weight and mammal to bediagnosed, the particular type of liposome to be employed, and mostimportantly, the particular area of the patient to be scanned.

The following Examples are merely illustrative of the present inventionand should not be considered as limiting the scope of the invention inany way. These examples and equivalents thereof will become moreapparent to those versed in the art in light of the present disclosure,and the accompanying claims.

EXAMPLES Example 1

Egg phosphatidylcholine, 1 gram, was suspended in 100 cc ofphysiological saline at room temperature to form a dispersion ofmultilamellar liposome vesicles. The liposomes were then placed in thevessel of FIG. 5. The outlet valve on the vessel was then sealed and thesystem was pressurized with between 30 to 50 psi CO₂ gas. The suspensionis then emptied into a flask and the non-encapsulated CO₂ gas wasallowed to escape. CO₂ gas entrapped within the vesicles remainedentrapped. The gas filled vesicles surprisingly did not float, but weredistributed evenly in solution. The resultant gas filled liposomes werefound to be intensely echogenic on ultrasound.

Example 2

Vesicles were also formed as described in Example 1, except that vesicleformation was carried out in the presence of bicarbonate and theionophore A23187 resulting in bicarbonate encapsulated liposomescontacting that ionophore. Acid was added to the external aqueous phasein order to lower the pH within the vesicles. The bicarbonate entrappedwithin the vesicles was found to form CO₂ gas and water.

Examples 3-18

A. Liposome Preparation

To prepare multilamellar vesicles (MLV's) pure egg phosphatidylcholine(EPC) obtained from Avanti Polar Lipids (Birmingham, Alabama) wassuspended in phosphate buffered saline (PBS) and swirled by hand. Inother cases vesicles of defined size were prepared by a process ofextrusion with or without a preceding freeze-thaw process. Two differentlipid mixtures were tested, either pure EPC or a mixture of 80 molepercent EPC with 20 mole percent cholesterol. Typically, for theEPC/cholesterol vesicles 3.6 mmol (2.83 g) of EPC and 1.2 mmol (0.48 g)of cholesterol were dissolved together, in a minimum volume ofchloroform, in a 250-ml round-bottom flask. The chloroform was removedby rotary evaporation under reduced pressure to leave a thin film on thewalls of the flask; the contents were then held under reduced pressure(less than 0.1 mm Hg) for at least 2 hours to remove residual solvent.For mixtures of pure EPC the step of suspension in chloroform wasomitted. For both pure EPC and the dried film of EPC/cholesterol, thelipid was dispersed by vigorous mixing in 20 cc of neutral pH phosphatebuffered saline (PBS).

Vesicles prepared both with and without freeze-thaw were synthesized.For vesicles subjected to freeze-thaw the multilamellar vesicles formedupon dispersion were transferred to cryovials and then quench frozen inliquid nitrogen. The cryovials were then placed in warm water until thelipid suspension had completely thawed. This cycle of freezing andthawing was repeated four more times. Both the vesicles subjected tofreeze-thaw and those which were not freeze-thawed were then sized byten passes under nitrogen pressure (approx. 100 psi) through two stacked2 micron filters (Nucleopore, Pleasanton, Calif.) using the extruderdevice (Lipex Biomembranes, Vancouver, British Columbia, Canada). Aportion of this sized preparation was then passed ten times through twostacked 0.4 micron filters, a portion of this was then passed tenadditional times through 0.2 micron filters, and finally a portion ofthis was passed ten times through 0.030 micron filters. The above wasconducted for the EPC/cholesterol vesicles but filter sizes of only 0.4and 0.2 microns were tested for the vesicles composed of pure EPC. Thesizes of the resultant vesicles were determined by quasi elastic lightscattering with a Nicomp (Goleta, Calif.) Model 270 particle sizeroperating at 634.2 nm by standard cumulants analysis.

B. Pressurization

Pressurization of the liposome solutions and controls of PBS wasaccomplished with a soda seltzer bottle using carbon dioxide cartridges(FIG. 4). In all experiments for dilution of liposomes and as controlsonly degassed solutions of PBS were used. All liquids were degassedimmediately prior to use. Degassification of solutions was accomplishedby reducing pressure under vacuum. In pressurization of all solutions 50cc of liquid was poured into the seltzer bottle and the system thensealed. Pressurization was then accomplished by engaging the CO₂cartridge with the bottle. After 1 minute the pressure was released fromthe bottle and the solution poured from the bottle. The seltzer bottlewas equipped with a pressure gauge and the pressure was measured duringeach experiment. Solutions which were exposed to gassification includedvarious dilutions of the different sizes of liposomes and pure PBS.

C. Ultrasound Imaging

Ultrasound Imaging was performed with an Acuson 128 scanner (Milpitas,Calif.) using a 7.5 megahertz linear array transducer. Post-processingfunction was linear with pre-processing set at 0 and persistence at 2.Phantom solutions (controls and liposomal agents) at variableconcentrations and constant volumes and depth were scanned at 30 to 60dB gain settings within thin plastic containers. Multifocal zones with adecreased frame rate were used for most images. For quantitativemeasurements a 1 cm circle was positioned on the images at a position 2cm below the transducer and the number of reflections within the circlewas counted. At the time of measurement of acoustic reflections theultrasonographer was blinded to information as to which contrast agentor control was being studied.

D. Results

The sizes of the resulting vesicles and their echogenicity on ultrasoundare shown in Tables I and II. The size of the vesicles is controlled bythe filters used in the extrusion process. For the MLV's preparedwithout extrusion the size range is quite variable. For those vesicleswhich underwent the extrusion process the size range is comparativelynarrow.

In the ultrasound imaging experiments of the phantoms containing thedifferent contrast agents large reflections were seen at 30 dB for thefirst minute or two after the ultrasound contrast agents werepressurized and then poured into the plastic phantom dishes. These largereflections decayed quickly however and were not visible after severalminutes. The reflections which persisted after the first 2 to 3 minuteswere of much finer size and more regular in appearance.

The number of reflections counted within a 1 cm diameter circle overtime for the different solutions is shown in Tables I and II. The 0.4micron vesicles composed of pure EPC synthesized by extrusion butwithout freeze-thaw had the greatest echogenicity after exposure topressurization and echogenicity was sustained for 2 hours after thepressurization process. By comparison the phantom containing a similarconcentration of the same vesicles without exposure to pressurizationhad no internal echoes at all at 30 minutes after they were poured intothe phantom. MLV's synthesized of pure EPC had the next highestechogenicity. All of the vesicles which contained cholesterol had lowerechogenicity than the vesicles composed of pure EPC. The controlsolution of PBS exposed to pressurization but not containing liposomeshad echoes during the first minute or two but these echoes decayedrapidly to zero after several minutes (Table II).

The greatest echogenicity was seen in 0.4 micron vesicles composed ofpure EPC which were extruded but not freeze-thawed. Vesicles of the samesize which were freeze-thawed had less echogenicity. Vesicles of thissize subjected to freeze-thaw will be unilamellar whereas those notsubjected to freeze-thaw of this size will be oligolamellar.

                  TABLE I*                                                        ______________________________________                                        Number of Reflections at 60 dB                                                          Time in minutes                                                     Example     5     10    20   30  45    60   120                               ______________________________________                                         3 (0.4μ, no FT)                                                                       --    --    --   95  89    116  109                                4 (0.4μ + FT)                                                                         --    --    --   52  69    --   --                                 5 (0.2μ + FT)                                                                         --    --    --   26  52    --   --                                 6 (MLV's)  --    --    58   72  --    --   --                                 7 (Ex. 6 at 94 psi)                                                                      --    60    69   --  --    --   --                                 8 (MLV's, no gas)                                                                        84    32    10   --  --    --   --                                 9 (Ex. 3, no gas)                                                                        18    --    --    0  --    --   --                                10 (PBS)     0    --    --   --  --    --   --                                11 (PBS + gas)                                                                            12    10     3   --  --    --   --                                ______________________________________                                         *Data above from counting number of reflections within 1 cm diameter          circle positioned 2 cm from transducer on images obtained by scanning 400     cc solutions of ultrasound contrast agents with 7.5 mHz linear array          transducer. The liposomes in Examples 3 through 6 were pressurized with 5     to 54 psi CO.sub.2 gas. The liposomes in Examples 3, 4 and 5 were extrude     10 times through filters as specified, in Examples 4 and 5, these             liposomes were then exposed to 5 cycles of freezethaw, and in Example 3,      these liposomes were not freezethawed. The liposomes in Example 6 involve     multilamellar vesicles (MLV's) prepared by simple mixing of egg               phosphatidylcholine (EPC). The liposomes in Example 7 were exposed to         higher pressure of 94 psi. The liposomes in Example 8 and 9 are control       samples of vesicles, with those in Example 8 involving MLV's and those in     Example 9 being 0.4μ vesicles (no freezethaw) without exposure to gas.     In Example 10, the sample is phosphate buffered normal saline (PBS)           without exposure to gas. In all vesicle preparations final lipid              concentration is 1.25 micromoles/ml. The notation (--) indicates that the     number of reflections was not measured.                                  

                  TABLE II*                                                       ______________________________________                                        Number of Reflections at 60 dB                                                               Time in minutes                                                Example           1     2     3  10  15  20  30                               ______________________________________                                        12 (0.2μ)     22    30    33  --  --  --  27                               13 (MLV's)       40    27    34  --  --  --  48                               14 (2.0μ EPC/Chol.)                                                                         --    18    --  --  15  15  --                               15 (MLV's 50 nm EPC/Chol.)                                                                     12    23    13  --  --  16  15                               16 (0.4μ EPC/Chol.)                                                                         15    22    --  --  11  --  15                               17 (PBS + gas)   15     7     6  10   3   0   0                               18 (PBS, no gas) 23    25     4  --  --   0  --                               ______________________________________                                         *Lipid concentration in the above is 0.225 micromoles of lipid per ml. Th     liposomes in Examples 12 and 13 are pure EPC, the liposomes in Example 13     are MLV's as in Table I, and the liposomes in Example 12 are a dilute         version of those employed in Example 5 from Table I. Examples 12, 14, 15      and 16 were produced by extrusion through filter pore sizes as specified      and Examples 14, 15 and 16 contain 80% EPC/20% Cholesterol.              

Having described the invention above, it will be obvious to one skilledin the art that various parameters such as liposome size and membranecomposition are selected to achieve the desired effect in terms ofbiodistribution and imaging.

Various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

What is claimed is:
 1. A method of providing an image of an internalregion of a patient comprising:(a) administering to the patient aliposome having encapsulated therein a solid contrast enhancing agentselected from the group consisting of magnetite and solid iodineparticles, or a liquid contrast enhancing agent selected from the groupconsisting of solubilized iodinated compounds; and (b) scanning thepatient using ultrasonic imaging to obtain visible images of the region.2. The method according to claim 1 wherein said liposomes areadministered intravascularly, and the vesicles have a mean outerdiameter of between about 30 nanometers and about 2 microns.
 3. Themethod according to claim 2; wherein the patient is scanned in the areaof the patient's heart.
 4. The method according to claim 2 wherein thepatient is scanned in the area of the patient's arterial system.
 5. Themethod according to claim 2 wherein the patient is scanned in the areaof the patient's venous system.
 6. The method according to claim 2wherein the patient is scanned in the area of the patient's liver,spleen and kidney.
 7. The method according to claim 1 wherein theliposomes are administered other than intravascularly and have a meanouter diameter between about 2 microns and about 10 microns.
 8. Themethod of claim 1 wherein the liposomes are comprised of lipids ofeither natural or synthetic origin selected from the group consisting offatty acids, lysolipids, dipalmitoylphosphatidylcholine,phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol,phosphatidylserine, phosphatidic acid, phosphatidylinositol,sphingomyelin, lysolipids, cholesterol, cholesterol hemisuccinate,glycosphingolipids, glycolipids, glucolipids, sulphatides, andtocopherol hemisuccinate.
 9. A method for diagnosing the presence ofdiseased tissue in a patient comprising:(a) administering to the patienta liposome having encapsulated therein a solid contrast enhancing agentselected from the group consisting of magnetite and solid iodineparticles, or a liquid contrast enhancing agent selected from the groupconsisting of solubilized iodinated compounds; and (b) scanning thepatient using ultrasonic imaging to obtain visible images of anydiseased tissue in the patient.
 10. A method according to claim 1wherein said contrast enhancing agent is a solid selected from the groupconsisting of magnetite and solid iodine particles.
 11. A methodaccording to claim 11 wherein said solid iodine particles are selectedfrom the group consisting of iodipamide ethyl ester and metricate.
 12. Amethod according to claim 11 wherein said solid iodine particles areiodipamide ethyl ester.
 13. A method according to claim 1 wherein saidcontrast enhancing agent is a liquid selected from the group consistingof solubilized iodinated compounds.
 14. A method according to claim 9wherein said liposomes are administered intravascularly, and thevesicles have a mean outer diameter of between about 30 nanometers andabout 2 microns.
 15. A method according to claim 14 wherein the patientis scanned in the area of the patient's heart.
 16. A method according toclaim 14 wherein the patient is scanned in the area of the patient'sarterial system.
 17. A method according to claim 14 wherein the patientis scanned in the area of the patient's venous system.
 18. A methodaccording to claim 14 wherein the patient is scanned in the area of thepatient's liver, spleen and kidney.
 19. A method according to claim 9wherein the liposomes are administered other than intravascularly andhave a mean outer diameter between about 2 microns and about 10 microns.20. A method of claim 9 wherein the liposome is comprised of lipidselected from the group consisting of fatty acids, lysolipids,dipalmitoylphosphatidylcholine, phosphatidylcholine,phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine,phosphatidic acid, phosphatidylinositol, sphingomyelin, lysolipids,cholesterol, cholesterol hemisuccinate, glycosphingolipids, glycolipids,glucolipids, sulphatides, and tocopherol hemisuccinate.
 21. A methodaccording to claim 9 wherein said contrast enhancing agent is a solidselected from the group consisting of magnetite and solid iodineparticles.
 22. A method according to claim 20 wherein said solid iodineparticles are selected from the group consisting of iodipamide ethylester and metricate.
 23. A method according to claim 22 wherein saidsolid iodine particles are iodipamide ethyl ester.
 24. A methodaccording to claim 9 wherein said contrast enhancing agent is a liquidselected from the group consisting of solubilized iodinated compounds.